The overall objectives are to develop nuclear magnetic resonance (NMR) techniques and use them in concert with other experimental approaches to elucidate the molecular structure and physiologic functions of selected membrane-targeting proteins involved in phototransduction in vision and other signal transduction processes. During the next five years, we will use NMR, fluorescence, microcalorimetry, spin-label EPR, x-ray crystallography, and computational analysis to delineate the structure, dynamics and mechanisms of a family of neuronal calcium sensor proteins (calcium-myristoyl switches) that serve as membrane-targeting regulators in calcium signaling and are linked to retinal and neurological diseases. Our studies will focus on retinal recoverin, involved in cancer-associated retinopathy;guanylate cyclase activating proteins (GCAPs), linked to autosomal dominant cone dystrophy;and calcium binding protein-4 (CaBP4), implicated in autosomal recessive night blindness. By continuing our intensive study of retinal recoverin and the GCAP proteins and by broadening its scope to encompass neuronal homologs and protein targets, we hope to gain an atomic-level understanding of how calcium sensor proteins operate in signal transduction and disease processes. In particular, we want to understand how covalently attached myristoyl groups work in concert with calcium-binding sites and target proteins to guide this family of proteins to specific membrane-bound targets. The specific aims are 3-fold: (1) Determine the structure of the activator forms of GCAP1 (Ca2+ free state and constitutively active mutants) and probe their structural interactions with retinal guanylate cyclase. (2) Determine the atomic-level structures of Ca2+-myristoyl switch proteins and their target proteins assembled on lipid bilayer membranes using novel spin-label EPR and solid-state NMR techniques combined with computational molecular dynamics simulations. (3) Determine the atomic-level structures of the retinal calcium sensor protein (CaBP4) and elucidate its structural interaction and regulation of retinal Ca2+ channels (CaV1.4) at the rod and cone synapse implicated in retinal diseases. PUBLIC HEALTH RELEVANCE: Calcium ion (Ca2+) controls the excitability of light-sensitive rod and cone cells in the retina and defects in light-dependent calcium signaling are linked to retinal degenerative diseases. The goal of our research is to elucidate the molecular structure and mechanisms of calcium sensor proteins that regulate light-adaptation and disease processes during visual excitation.